Structural and conformational changes in models of β-lactoglobulin modified by Michael addition of 1,2-benzoquinones using molecular dynamics and metadynamics simulations

Food proteins react with o -quinones (oxidized polyphenols), and the resulting protein-polyphenol Michael adducts can alter the protein structure leading to changes in functional properties of food proteins. The present work investigated structural changes in models of protein-polyphenol Michael adducts (exemplified with β-lactoglobulin (β-LG) covalently modified with 4-methylcatechol (4MC), a model polyphenol similar to the B ring of flavonoids) using molecular dynamics (MD) and metadynamics (MTD) simulations. Based on experimentally identified 4MC modified sites in β-LG, β-LG-4MC model adducts (hereafter referred to as β-LQ) were designed with 19 site-specific 4MC modifications (β-LQ19) and with the four most abundant 4MC modifications (β-LQ4). The unliganded structure of β-LG was used as control. 4MC modifications in β-LQ model adducts compared with β-LG resulted in increased protein flexibility, however the tertiary structures remained unaffected by the modifications during 400 ns MD simulations. Both MD and MTD (with secondary structure as independent variable) data indicated that the α-helix structure was more susceptible to loss compared to the β-sheet structure in β-LQ model adducts, as opposed to β-LG. The metastable states determined with MTD simulations (with root mean squared deviations as independent variable) showed unfolding in thermally sensitive regions (amino acid residues 57 to 70) in β-LQ19. The structural changes (loss of α-helix and unfolding) exhibited by β-LQ19 explain the experimentally determined reduced denaturation temperature in β-LQ compared with β-LG. • Structures of β-lactoglobulin(β-LG)-4-methylcatechol(4MC) adducts were characterized • β-LG-4MC model adducts were designed and simulated • Molecular dynamics simulations showed well conserved tertiary structure in adducts • Metadynamics simulations revealed 4MC induced unfolding in β-LG • Simulations explained reduced denaturation temperature in β-LG-4MC adducts